Recombinant Human LIPA HA-tag His-tag Protein, CF

Recombinant Human LIPA HA-tag His-tag (Catalog # 11633-LA) is measured by its ability to cleave a fluorogenic peptide substrate, 4-Methylumbelliferyl oleate (4-MUO).

2 μg/lane of Recombinant Human LIPA HA-tag His-tag (Catalog # 11633-LA) was resolved with SDS-PAGE under reducing (R) and non-reducing (NR) conditions and visualized by Coomassie® Blue staining, showing bands at 55-67 kDa, under reducing conditions.

• Reactivity: Hu
• Applications: Enzyme Activity
• Format: Carrier-Free

Recombinant Human LIPA HA-tag His-tag Protein, CF Summary

• Details of Functionality: Measured by its ability to cleave a fluorogenic substrate, 4-Methylumbelliferyl oleate (4-MUO).The specific activity is >7000 pmol/min/μg, as measured under the described conditions.
• Source: Chinese Hamster Ovary cell line, CHO-derived human LIPA protein
  Gly24-Gln399 with N-terminal HA (YPYDVPDYA) and 6-His tags
• Accession #: P38571.2
• N-terminal Sequence: Tyr
• Protein/Peptide Type: Recombinant Enzymes
• Purity: >95%, by SDS-PAGE visualized with Silver Staining and quantitative densitometry by Coomassie® Blue Staining
• Endotoxin Note: <0.10 EU per 1 μg of the protein by the LAL method.

Applications/Dilutions

• Dilutions:
  • Enzyme Activity
• Theoretical MW: 45 kDa.
  Disclaimer note: The observed molecular weight of the protein may vary from the listed predicted molecular weight due to post translational modifications, post translation cleavages, relative charges, and other experimental factors.
• SDS-PAGE: 55-67 kDa, under reducing conditions

Packaging, Storage & Formulations

• Storage: Use a manual defrost freezer and avoid repeated freeze-thaw cycles.
  • 6 months from date of receipt, -70 °C as supplied.
  • 3 months, -70 °C under sterile conditions after opening.
• Buffer: Supplied as a 0.2 μm filtered solution in MES, NaCl and TCEP.
• Purity: >95%, by SDS-PAGE visualized with Silver Staining and quantitative densitometry by Coomassie® Blue Staining
• Assay Procedure:
  • Assay Buffer: 25 mM MES, 1% Triton X-100, pH 5.5
  • Recombinant Human LIPA HA-tag His-tag (rhLIPA) (Catalog # 11633-LA)
  • Substrate: 4-Methylumbelliferyl oleate (4-MUO), 100 mM stock in DMSO
  • Black 96-well Plate
  • Plate Reader with Fluorescence Read Capability
  1. Dilute rhLIPA to 2 µg/mL in Assay Buffer.
  2. Dilute Substrate to 1.2 mM in Assay Buffer.
  3. Load into a plate 50 µL of 2 µg/mL rhLIPA and start the reaction by adding 50 µL of 1.2 mM Substrate. Include a Substrate Blank containing 50 µL of Assay Buffer and 50 µL of 1.2 mM Substrate.
  4. Read at excitation and emission wavelengths of 365 nm and 445 nm (top read), respectively, in kinetic mode for 5 minutes.
  5. Calculate specific activity:

  Specific Activity (pmol/min/µg) = (Adjusted Vmax* (RFU/min) x Conversion Factor** (pmol/RFU)) / (amount of enzyme (µg))

  *Adjusted for Substrate Blank
  **Derived using a fluorescent standard 4-Methylumbelliferone
  Per Well:
  • rhLIPA: 0.1 µg
  • Substrate: 600 µM

Notes

This product is produced by and ships from R&D Systems, Inc., a Bio-Techne brand.

Alternate Names for Recombinant Human LIPA HA-tag His-tag Protein, CF

• Acid cholesteryl ester hydrolase
• CESD
• Cholesteryl esterase
• EC 3.1.1
• EC 3.1.1.13
• LALCESDcholesterol ester hydrolase
• LIPA
• Lipase A
• lipase A, lysosomal acid, cholesterol esterase
• lysosomal acid lipase
• lysosomal acid lipase/cholesteryl ester hydrolase
• Sterol esterase

Background

Recombinant human Lysosomal acid lipase (LAL) or Lipase A (LIPA), also known as acid cholesteryl ester hydrolase, is a 378 residue mature, glycosylated, lysosomal serine hydrolase of a family of three mammalian acid lipases. Mature LIPA contains a globular core domain typical of a/b hydrolase-fold family members and a cap domain. Beneath the cap region, the core domain contains the catalytic triad surrounded by a hydrophobic surface that promotes lipid substrate binding (1). Except for erythrocytes, LIPA is broadly expressed in cells (1) and is found in the lysozyme where it is required for hydrolysis of cholesteryl esters and triglycerides from low density lipoproteins (LDL) to generate fatty acids and cholesterol (2). LIPA deficiency from over 40 reported loss-of-function mutations leads to accumulation of cholesterol and triglyceride in the lysosome with resulting dysfunctional cholesterol homeostasis (1, 3). Lysosomal acid deficiency (LAL-D) is an autosomal recessive lysosomal storage disease with two phenotypes including early-onset Wolman disease (WD) and later-onset cholesteryl ester storage disease (CESD) (3, 4). Supplementation of LIPA through enzyme replacement therapy has been shown to be effective and is the standard therapy for LAL-deficiency although additional treatments are being explored (3, 4-6). Due to its central role in lipid regulation, altered function and variants of LIPA have also been implicated in development of atherosclerotic disease and coronary artery disease (1, 3, 7, 8). LIPA has additionally been reported as a therapeutic target for cancer and solid tumors through its role in induction of ER stress (9), its association with specific binding partner of interest (10), and its ability to modulate lipid metabolism in cancer cells (11).

1. Rajamohan, F. et al. (2020) J. Lipid Res. 61:1192.
2. Anderson, R.A. and G.N. Sando (1991) J. Biol. Chem. 266:22479.
3. Maciejko, J.J. (2017) Am. J. Cardiovasc. Drugs 17:217.
4. Korbelius, M. et al. (2023) Trends Mol. Med. 29:425.
5. Burton, B.K. et al. (2015) N. Engl. J. Med. 373:1010.
6. Rader, D.J. (2015) N. Engl. J. Med. 373:1071.
7. Wild, P.S. et al. (2011) Circ. Cardiovasc. Genet. 4:403.
8. Li, F. and H. Zhang (2019) Arterioscler. Thromb. Vasc. Biol. 39:850.
9. Liu, X. et al. (2022) Nat. Cancer 3:866.
10. Collier, A.B. (2024) Cancers 16:500.
11. Jin, Z. et al. (2024) Cancer Res. Commun. 4:2242.

Bioinformatics

• Uniprot:
  • P38571.2()